title: Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.
abstract: Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recognition elements. We have constructed a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85 degrees C for 1 h, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long-term storage and in-line monitoring require very rugged yet highly specific recognition elements.
authors.affiliation: Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, USA. ellen.goldman@nrl.navy.mil
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.forename: Jerry
authors.initials: J
authors.lastname: Conway
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Sherwood
authors.forename: Melissa
authors.initials: M
authors.lastname: Fech
authors.forename: BaoHan
authors.initials: B
authors.lastname: Vo
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.forename: Andrew
authors.initials: A
authors.lastname: Hayhurst
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000276:immunology*; D002161:Camelids, New World / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D008969:Molecular Sequence Data; D055550:Protein Stability; D012703:Serotyping; D012995:Solubility; D013696:Temperature; D014121:Toxoids / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D014121:Toxoids; D019274:Botulinum Toxins, Type A